Adjuvant activities of B pentamers of LT-IIa and LT-IIb enterotoxin
The present invention provides a method for enhancing an immunological response to an antigen. The method comprises administering to an individual a composition comprising an antigen and an isolated LT-IIa-B pentamer or a mutant thereof, or an isolated LT-IIb-B pentamer or a mutant thereof. The selected LT-II-B pentamer acts as an adjuvant to enhance the immunological response to the co-administered antigen.
This application claims priority to U.S. provisional patent application Ser. No. 60/653,235, filed Feb. 15, 2005, the disclosure of which is incorporated herein by reference.
This work was supported by Grant nos. DE13833, DE015254, DE06746 from the National Institutes of Health. The Government has certain rights in the invention.
FIELD OF THE INVENTIONThe present invention relates generally to the field of adjuvants and more particularly to adjuvant activities of B pentamers of LT-IIa and LT-IIb enterotoxin.
DISCUSSION OF RELATED ARTSince mucosal surfaces represent the major entry route of many microbial pathogens, it is important that prospective vaccines stimulate appropriate immune response at these sites.
However, the mucosal immune system usually requires the aid of immune stimulating agents (i.e., adjuvants) to generate robust immunity and long-lived memory responses to an antigen. The type I heat-labile enterotoxins produced by Vibrio cholerae and Escherichia coli (CT and LT-I, respectively) have been extensively characterized as mucosal adjuvants in a variety of animals (Harandi, A. M., et al., 2003, Curr. Opin. Investig. Drugs 4:156-161). The immunomodulatory activities of a second class of heat-labile enterotoxins of E. coli have also been described. This second class consists of LT-IIa and LT-IIb, two heat-labile enterotoxins from E. coli which can be distinguished from LT-I by a variety of antigenic and genetic differences (Guth, B. E., et al., 1986, Infect Immun 54:587-589, Guth, B. E., et al., 1986, Infect Immun 54:529-536). Murine experiments demonstrated that certain immunomodulatory activities of LT-IIa and LT-IIb are equivalent or greater than those of CT (Connell, T. D., et al., 1998, Immunology Letters 62:117-120, Martin, M., et al., 2000, Infect. Immun. 68:281-287).
The E. coli heat-labile enterotoxins LT-I, LT-IIa, LT-IIb and CT belong to the AB5 superfamily of bacterial enterotoxins. Members of this superfamily are related in structure and function (Guth, B. E., et al., 1986, Infect Immun 54:587-589, Guth, B. E., et al., 1986, Infect Immun 54:529-536, Spangler, B. D., 1992, Microbiol. Rev. 56:622-47, van den Akker, F., et al., 1996, Structure 4:665-678). Each of these enterotoxins is an oligomeric protein composed of an A polypeptide which is noncovalently coupled to a pentameric array of B polypeptides. The A polypeptide is enzymatically active and upregulates adenylyl cyclase by catalyzing the ADP-ribosylation of the Gsα regulatory protein. This modification of Gsα promotes accumulation of intracellular cAMP which indirectly induces the intoxicated cell to secrete chloride ions and likely modulates other processes for which cAMP is a signaling molecule (Cassel, D., et al., 1977, Proc. Natl. Acad. Sci. U S A 74:3307-3311, Holmes, R. K., et al., 1995,. Bacterial Toxins and Virulance Factors in Disease, vol. 8. Marcel Dekker, Inc., New York, Moss, J., et al., 1979, J. Biol. Chem. 254:11993-11999, Moss, J., et al., 1979, Annu. Rev. Biochem. 48:581-600, Moss, J., et al., 1977, J. Biol. Chem. 252:2455-2457), and which is believed to cause the dehydrating symptoms associated with infection by cholera and certain strains of E. Coli.
The B pentamer mediates binding of LT-IIa, LT-IIb, CT, and LT-I to gangliosides, a heterogeneous family of glycolipids located on the surface of mammalian cells (Sonnino, S., et al., 1986, Chem. Phys. Lipids 42:3-26). CT and LT-I bind with high affinity to GM1 and with lower affinity to ganglioside GD1b; LT-IIa-binds specifically, in descending order of avidity, to gangliosides GD1b, GM1, GT1b, GQ1b, GD2, GD1a and GM3; LT-IIb-binds most avidly to GD1a, and to GM2 and GM3 with much lower affinities (Fukuta, S., et al., 1988, Infect. Immun. 56:1748-1753).
LT-IIa, LT-IIb, CT, and LT-I are potent mucosal and systemic adjuvants capable of eliciting strong immune responses to themselves and to unrelated co-administered antigens (Connell, T. D., et al., 1998, Immunology Letters 62:117-120, (Elson, C. O., 1989, Curr. Top. Microbiol. Immunol. 146:29-33, Martin, M., et al., 2000, Infect. Immun. 68:281-287, McCluskie, M. J., et al., 2001, Vaccine 19:3759-3768, Plant, A., et al., 2004, Curr. Top. Med. Chem. 4:509-519, Sougioultzis, S., et al., 2002, Vaccine 21:194-201). However, use of these enterotoxins as mucosal adjuvants in human vaccines has been inhibited by the toxic activity mediated by their A subunits. Thus, there is an ongoing need for improved enterotoxin-based compositions that can be safely used as adjuvants.
SUMMARY OF THE INVENTIONThe present invention provides a method for enhancing an immunological response to an antigen. The method comprises administering to an individual a composition comprising an i) antigen, and ii) an isolated LT-IIa-B pentamer or a mutant thereof, or an isolated LT-IIb-B pentamer or a mutant thereof, whereby administration of the B pentamer of ii) acts as an adjuvant to enhance the immunological response to the antigen of i).
In the present invention, it was unexpectedly observed that compositions comprising B pentamers (and not their respective A subunits) can enhance an immunological response to an antigen, and that the immunological response is distinct from the immunological response enhanced by intact LT-II holotoxins to the same antigen. In particular, the B pentamers effectively induce proinflammatory cytokine release, but the holotoxins are ineffective at inducing proinflammatory cytokine release under the same experimental conditions. Further, the LT-II holotoxins, but not the B pentamers, downregulate proinflammatory signals and upregulate cytokines with anti-inflammatory properties, and thus may antagonize the distinct immunomodulatory effects of the B pentamers. Further, intact holotoxins, while also exhibiting IgA and IgG adjuvant activity, induced a substantial increase in cAMP production in vitro. In contrast, while the B pentamers also exhibited adjuvant activity for IgA and IgG, significantly less cAMP was produced by cells treated with the B pentamers alone. Therefore, compositions comprising B pentamers which have been isolated from their A subunits or produced recombinantly may be useful for enhancing an immune response to an antigen without eliciting unwanted side effects associated with the use of intact holotoxins. Further, certain B pentamer mutations result in altered or reduced receptor binding, which may reduce their capacity to participate in retrograde trafficking through the olfactory nerve.
BRIEF DESCRIPTION OF THE FIGURES
The present invention provides a method for enhancing an immunological response to an antigen. The method comprises administering to an individual an effective amount of a composition comprising an antigen and an isolated wild type B pentamer or an isolated mutant B pentamer of the E. coli heat-labile LT-IIa or LT-IIb holotoxins, whereby administration of the isolated B pentamer elicits an adjuvant effect to enhance the immunological response to the antigen.
As used herein, the term “isolated B pentamer” refers to a B pentamer that is not in association with an A subunit. Therefore, an isolated B pentamer may be a B pentamer that has either been biochemically separated from its A subunit, or a B pentamer that has been produced recombinantly.
Thus, compositions comprising either isolated wild type or isolated mutant B pentamers can be utilized in the method of the invention. When mutant B pentamers are used, they may be mutants that abrogate or substantially reduce binding to ganglioside receptors. Further, data presented herein demonstrates that the wild type B pentamers induce significantly less of at least one deleterious biochemical intermediate known to be associated with the symptoms of enterotoxin intoxication. Moreover, administration of either wild type or mutant B pentamers induces unexpectedly distinct and potentially beneficial immunological effects as compared to administration of the respective intact holotoxins.
In more detail, B pentamers of LT-IIa and LT-IIb, but not their respective holotoxins, are demonstrated herein to effectively induce proinflammatory cytokine release from human cells. In contrast, the intact LT-IIa and LT-IIb holotoxins, but not their respective B pentamers, are demonstrated to downregulate proinflammatory cytokines (TNF-α) or chemokines (IL-8) and upregulate cytokines with anti-inflammatory (IL-10) properties, indicating the B pentamers may be superior to the holotoxins in stimulating an adaptive immune response. Data presented herein also strongly implicates the Toll-Like Receptors in cellular activation by the B pentamers. In contrast, the LT-IIa and LT-IIb holotoxins do not significantly activate TLR-expressing cells. Thus, isolated B pentamers unexpectedly have an immunological effect that is not exerted by intact holotoxins.
It is additionally demonstrated herein that mucosal (nasal) administration of isolated LT-IIa-B pentamers or LT-IIb-B pentamers (as well as their respective intact holotoxins) in a mouse model results in strong adjuvant activity at mucosal surfaces against a co-administered antigen. Significantly, an augmented adjuvant response was also induced at distal mucosa (vaginal secretions) by the B pentamers and the intact holotoxins. Further, both isolated B pentamers and the holotoxins exhibit the capacity to augment strong antigen-specific IgG responses in serum when employed as a mucosal adjuvant. However, while the holotoxins induced a large increase in cAMP production in vitro, much less cAMP production was induced by use of the B pentamers alone. Therefore, administration of compositions comprising isolated LT IIa-B pentamers, LT IIb-B pentamers, or mutants thereof, may have important and heretofore unrecognized advantages over their respective intact holotoxins.
Isolated B pentamers of LT-IIa or LT-IIb, and mutants thereof, can be obtained by standard recombinant molecular biology techniques. In this regard, intact holotoxins can be extracted from E. coli cultures and the B pentamers biochemically separated from the A subunits. Alternatively, suitable DNA cloning and mutagenesis methods, as well as procedures for expressing and purifying recombinant proteins are known. (See, for example, (Sambrook et al., 2001, Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York, N.Y.).
In general, to obtain wild type B pentamers, E. coli genomic DNA can be obtained from an E. coli culture according to standard methods. The DNA encoding the B pentamers can be amplified from the genomic DNA, such as by the polymerase chain reaction, and the amplification products can be cloned into a suitable vector for expression and purification of the B pentamers. (The B pentamers are believed to spontaneously pentamerize in solution under physiological conditions.) The B pentamers can be subsequently extracted and purified from the culture according to standard techniques.
Similarly, DNA sequences encoding mutant B pentamers can be prepared using standard mutagenesis techniques. For this purpose, genomic E. coli DNA encoding wild type B pentaers can be amplified and isolated described above, and the desired mutations can be engineered into the B pentamer DNA coding sequences according to standard methodologies. The mutant B pentamer encoding DNA sequences can then be cloned into a suitable expression vector, expressed and purified from culture in the same manner as the wild type B pentamers.
In one embodiment, a mutant LT-IIa-B with a Thr to Ile substitution at position 34 (termed “LT-IIa-B(T34I)”) is provided.
In another embodiment, a mutant LT-IIb-B with a Thr to Ile substitution at position 13 (termed “LT-IIb-B(T13I)” is provided.
In additional embodiments, suitable B subunit mutants include, for LT-IIa (Connell et al., Infection and Immunity, 60:63-70, 1992), substitutions of I, P, G, N, L, R for T at the 13th position; substitutions of I, P, D, H, N for T at the 14th position; substitutions of A, G, M, H, L, R, Q for T at the 34th position. For B sununits of LT-IIb (Connell et al., Molecular Microbiology 16:21-31, 1995), substitutions of I, K, N for T at the 13th position; and substitutions of I, N, R, M, K for T at the 14th position.
For use as adjuvants, suitably purified wild type or mutant B pentamers can be combined with standard pharmaceutical carriers. Acceptable pharmaceutical carriers for use with proteins and co-administered antigens are described in Remington's Pharmaceutical Sciences (18th Edition, A. R. Gennaro et al. Eds., Mack Publishing Co., Easton, Pa., 1990).
In one embodiment, the antigen AgI/II, which is known to be poorly antigenic, is obtained from Streptococcus mutans cultures or is prepared using recombinant techniques. However, those skilled in the art will recognize that the method of the invention can be used to enhance the immune response to any antigen. Thus, the method can be used to enhance the immunogenicity of cancer vaccines, viral vaccines, bacterial vaccines or parasitic vaccines.
Further, in addition to being used as a co-mingled adjuvant, the B subunits can be used as carriers of antigens chemically coupled to the B pentamers to increase the immune response to the coupled antigen. This is particularly advantageous for mucosal routes of immunization to enhance the delivery of the antigen to the immune response tissues. Examples of antigens that may be coupled in this way include proteins, segments of proteins, polypeptides, peptides, and carbohydrates. Antigens can be coupled to isolated B subunits using a variety of conventional methods ways. For example, proteins, polypeptides, peptides, or carbohydrates can be chemically conjugated to enterotoxin B subunits by means of various well-known coupling agents and procedures, for example: glutaraldehyde, carbodiimide, bis-diazotized benzidine, maleimidobenzoyl-N-hydroxysuccinimide ester, N-succinimidyl-(3-[2-pyridyl]-dithio)propionate, cyanogen bromide, and periodate oxidation followed by Schiff base formation. Further, antigen peptides or polypeptides can be genetically fused to the N-terminus or C-terminus, or inserted into exposed loops of the B subunits, to obtain chimeric B pentamer/antigen molecules, by standard recombinant genetic DNA and protein expression technology.
Compositions comprising isolated B pentamers for use as adjuvants can be administered by any acceptable route. Suitable routes of administration include mucosal (e.g., intranasal, ocular, gastrointestinal, oral (including by inhalation), rectal and genitourinary tract), oral) and parenteral (e.g., intravascular, intramuscular, and subcutaneous injection). A preferred route of administration is intransal mucosal administration.
Those skilled in the art will recognize that the amount of B pentamers included in a pharmaceutical preparation will depend on a number of factors, such as the route of administration and the size and physical condition of the patient. The relative amounts of B pentamers in the pharmaceutical preparations can be adjusted according to known parameters. Further, the compositions comprising the B pentamers can be used in a single administration or in a series of administrations in a manner that will be apparent to those skilled in the art.
The following examples describe the various embodiments of this invention. These examples are illustrative and are not intended to be restrictive.
EXAMPLE 1This Example demonstrates engineering and purification of holotoxins and their B subunits. To engineer a His-tagged version of LT-IIa, a fragment encoding a portion of the A polypeptide and the B polypeptide was PCR amplified from pTDC400 (Connell, et al., 1992, Infect. Immun. 60:63-70) using the synthetic oligonucleotides 5′-GATGGGATCCTTGGTGTGCATGGAGAAA G-3′ (SEQ ID NO:1; BamHI site is underlined) and 5′-AAATAAACTAGTTTAGTGGTGG TGGTGGTGGTGTGACTCTCTATCTA ATTCCAT-3′ (SEQ ID NO:2; BcuI site is underlined; His codons are double underlined) as primers. PCR conditions were the following: denaturation at 95° C. for 45 s, annealing at 44° C for 45 s, and extension at 72° C. for 2 min, 30 cycles. After digestion with SacI and BcuI, the resulting PCR fragment was substituted for the SacI/BcuI fragment of pTDC200ΔS. This plasmid was derived from pTDC200 (Connell, et al., 1992, Infect. Immun. 60:63-70) upon removal of a redundant SacI restriction site by partial digestion with SacI, followed by blunting the digested site with Klenow fragment and religation with T4 DNA ligase. The plasmid encoding the LT-IIa holotoxin with a His-tagged B polypeptide was denoted pHN4.
To construct a recombinant plasmid encoding the His-tagged B polypeptide of LT-IIa, pHN4 was digested with SacI and BcuI. The obtained DNA fragment (encoding the B polypeptide) was inserted into pBluescript KSII+ (Stratagene, La Jolla, Calif.) at the SacI/BcuI sites to produce pHN15.
To engineer a His-tagged version of LT-IIb, a fragment carrying the genes for A and B polypeptides was PCR amplified from pTDC100 (Connell, T. D., et al., 1995, Mol. Microbiol. 16:21-31) using the synthetic oligonucleotides 5′-CGGGATCCATGCTCAGGTGAG-3′ (SEQ ID NO:3; BamHI site is underlined) and 5′-GGAATTCTTAGTGGTGGTGGTGGTGGTGTTCTGCCT CTAACTCGA-3′ (SEQ ID NO:4; EcoRI site is underlined; His codons are double underlined). PCR conditions were the following: denaturation at 95° C. for 45 s, annealing at 44° C for 45 s, and extension for 2 min, 30 cycles. After digestion with BamHI and EcoRI, the PCR fragment was ligated into pBluescript KSII+ at the BamHI/E coRI sites to produce pHN1, encoding LT-IIb holotoxin with a His-tagged B polypeptide.
Recombinant plasmid pHN16.1, encoding only the His-tagged B polypeptide of LT-IIb, was engineered by ligating the B-polypeptide-encoding XhoI/EcoRI fragment from pHN1 into pBluescript KSII+ at the XhoI and EcoRI sites.
To engineer a His-tagged version of the B subunit of CT (CTB), a fragment encoding a portion of the A polypeptide and the B polypeptide was PCR amplified from pSBR-CTΔA1 (Hajishengallis, G., et al., 1995, J. Immunol. 154:4322-4332) using the synthetic oligonucleotides 5′-TAAGAGCTCACTCGAGGCTTGGAGGGAAGAG-3′ (SEQ ID NO:5; SacI site is underlined) and 5′-TAACTAGTGCTGAGCTTAGTGGTGGTGGTGGTGGTGTATTTGCCATA CTAATTGC-3′ (SEQ ID NO:6; BcuI site is underlined; His codons-are double underlined) as primers. PCR conditions were the following: denaturation at 95° C. for 45 s, annealing at 44° C. for 45 s, and extension at 72° C. for 1 min, 30 cycles. After digestion with SacI and BcuI, the PCR fragment (corresponding to the B polypeptide) was inserted into pBluescript KSII+ at the SacI/BcuI sites to produce pHN14. CT was purchased from List Biological Laboratories, Campbell, Calif.
The sequence of the wild type LT-IIa-B polypeptide is as follows:
The first 23 amino acids represent the leader sequence and the sequence of the mature polypeptide is as follows:
The sequence of the wt LT-IIb-B polypeptide is:
The first 23 amino acids represent the leader sequence and the sequence of the mature polypeptide is as follows:
All plasmids for LT-II protein production were introduced into E. coli DH5αF'Kan (Life Technologies, Inc., Gaithersburg, Md.). Expression of recombinant holotoxin and B pentamers was induced by isopropyl-β-D-thiogalactoside, and the proteins were extracted from the periplasmic space by using polymyxin B treatment as previously described (Martin, M., et al., 2000, Infect. Immun. 68:281-287). Periplasmic protein extracts were precipitated by addition of ammonium sulfate to 60% saturation (390 g/liter). The precipitate was collected by centrifugation and was dissolved in phosphate-buffered saline (pH 7.4). The dissolved precipitate was dialyzed overnight in phosphate-buffered saline to remove ammonium sulfate, after which the recombinant proteins were purified by means of affinity chromatography using a His·Bind resin column (Novagen, Madison, Wis.) according to a protocol provided by the manufacturer. The eluted fraction was passed through a 0.45-μm-pore-size syringe filter and was further purified by means of gel filtration chromatography (Sephacryl-100; Pharmacia, Piskataway, N.J.) using an ÄKTA-FPLC (Pharmacia). The peak fractions were then concentrated using Vivaspin concentrators (Viva Science, Hanover, Germany). The purity of the recombinant proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All protein preparations were also analyzed by quantitative Limulus amebocyte lysate (LAL) assays (using kits from BioWhittaker, Walkersville, Md., or from Charles River Endosafe, Charleston, S.C.) to measure incidental endotoxin contamination. All holotoxin and B-pentamer preparations were essentially free of LPS (≧0.0064 ng/μg of protein). This was subsequently verified (see Results) in cytokine induction assays, the results of which were unaffected by the presence of the LPS inhibitor polymyxin B (10 jig/ml). Further evidence against contamination with heat-stable contaminants was obtained upon holotoxin or B-pentamer boiling, which destroyed their biological activity. The addition of His tag had no effect on the cytokine-inducing ability of the enterotoxins, as shown in preliminary experiments comparing non-His-tagged and His-tagged molecules (data not shown), which were thus subsequently used in the Examples herein.
Data presented in the Examples herein were evaluated by analysis of variance and the Dunnett multiple-comparison test using the InStat program (GraphPad Software, San Diego, Calif.). Statistical differences were considered significant at the level of P<0.05. Where appropriate, two-tailed t tests were also performed. Experiments were performed with triplicate samples and were performed twice or more to verify the results.
EXAMPLE 2This Example demonstrates the effects on cytokine induction of the LT-II holotoxins. Unlike CT or LT-I, LT-II toxins have not been previously examined for their capacity to induce cytokine release in monocytes/macrophages. This possibility was addressed in experiments using human monocytic THP-1 cells, which display a macrophage-like phenotype upon differentiation with phorbol myristate acetate (Auwerx, J., 1991, Experientia, 47:22-31, 16).
To perform THP-1 cell culture and cytokine induction assays, human monocytic THP-1 cells (ATCC TIB-202) were differentiated with 10 ng of phorbol myristate acetate/ml for 3 days in 96-well polystyrene culture plates at 37° C. in a humidified atmosphere containing 5% CO2. This cell line has been widely used as a model of human monocytes/macrophages (Auwerx, J., 1991, Experientia, 47:22-31). The culture medium consisted of RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies), 2 mM L-glutamine, 10 mM HEPES, 100 U of penicillin G/ml, 100 μg of streptomycin/ml, and 0.05 mM 2-mercaptoethanol. Differentiated THP-1 cells (1.5×105/well) were washed three times and were used in cytokine induction assays in the absence or presence of bacterial molecules as further specified herein. To determine the effect of toxins on cellular activation by LPS or other stimuli as indicated, the cells were pretreated for 1 hour with the toxins prior to stimulation. When toxins and LPS were added concomitantly to the cell cultures, either approach yielded similar data as further detailed in these Examples.
We examined induction of IL-1β, which possesses mucosal adjuvant properties, as well as cytokines that display proin-flammatory (TNF-α), chemotactic (IL-8), immunoenhancing (IL-6), or anti-inflammatory (IL-10) properties. LT-IIa and LT-IIb were tested at 2 μg/ml in comparison with an equal concentration of CT and with 10 ng of Ec-LPS/ml, a potent cytokine-inducing agonist. We found that LT-IIa and LT-IIb did not induce significant release of any of the cytokines tested (
This Example demonstrates the anti-inflammatory activity of the LT-II and CT holotoxins. We investigated whether LT-IIa and LT-IIb actively interfere with the proinflammatory activity of Ec-LPS, a strong TLR4 (Toll Like Receptor-4) agonist. Thus, induction of proinflammatory cytokines by Ec-LPS, a strong Toll-Like Receptor (TLR4) agonist was examined in THP-1 cells pretreated for 1 h with LT-IIa or LT-IIb enterotoxin or with CT. Other proinflammatory virulence factors that activate additional TLRs were also examined to determine whether inhibitory effects by the holotoxins could be extended to those molecules. Specifically, the effect of LPS from P. gingivalis, (Pg-LPS) which activates TLR2, and of recombinant P. gingivalis FimA, which activates TLR2 and TLR4 (Hajishengallis, G., et al., 2004, Infect. Immun. 72:1188-1191, Hajishengallis, G., et al., 2002, Infect. Immun. 70:6658-6664), were also determined.
The fimbrillin subunit (FimA) of Porphyromonas gingivalis fimbriae was purified by means of size-exclusion and anion-exchange chromatography from E. coli BL21 (DE3) transformed with the fimA gene of strain 381 (Hajishengallis, G., et al., 2002, Clin. Diagn. Lab. Immunol. 9:403-411). No LPS activity was detected in the FimA preparation by the LAL assay (BioWhittaker) following chromatography through agarose-immobilized polymyxin B (Detoxi-Gel; Pierce, Rockford, Ill.). LPS was purified from P. gingivalis 381 (Pg-LPS) or E. coli K235 (Ec-LPS) as previously described (Hajishengallis, G., et al., 2002, Infect. Immun. 70:6658-6664), yielding molecules that activate NF-κB exclusively through TLR2 or TLR4, respectively (Hajishengallis, G., et al., 2004, Infect. Immun. 72:1188-1191). The doses used for Ec-LPS, Pg-LPS, and FimA were chosen based on known parameters (Hajishengallis, G., et al., 2004, Infect. Immun. 72:1188-1191, Hajishengallis, G., et al., 2002, Infect. Immun. 70:6658-6664, Hajishengallis, G., et al., 2002, Clin. Diagn. Lab. Immunol. 9:403-411). None of these was found to affect the viability of the cells in the assays, as determined by trypan blue exclusion. Culture supernatants were collected after overnight incubation (16 h) and were stored at −80° C. until assayed. TNF-α, IL-1β, IL-6, IL-8, and IL-10 released into the culture medium were quantitated using enzyme-linked immunosorbent assay (ELISA) kits (purchased from eBioscience, San Diego, Calif., or Cell Sciences, Canton, Mass.) according to protocols recommended by the manufacturers.
Strikingly, all three holotoxins significantly (P<0.05) inhibited TNF-α induction by all three proinflammatory molecules, especially that by Ec-LPS (≧88% inhibition) (
To provide further evidence that the LT-II enterotoxins and CT interfere with inflammatory responses, we examined whether the enterotoxins also inhibit IL-8 induction by Ec-LPS. For this purpose, isolated B pentamers of each enterotoxin were examined in parallel with their respective holotoxins. The LT-II and CT holotoxins significantly (P<0.05) and potently inhibited IL-8 induced in response to a high concentration (1 μg/ml) of Ec-LPS (
The holotoxins and their B pentamers were also tested alone for their ability to induce IL-8 (
This Example demonstrates particular cytokine induction by the B subunits of LT-IIa and LT-IIb. To determine whether the B pentamers of LT-IIa and LT-IIb induced release of cytokines other than IL-8, THP-1 cells were treated with each B pentamer and the levels of TNF-α, IL-1β, and IL-6 were measured in the culture supernatants. All three cytokines were elicited by treatment with LT-IIb-B. In the case of TNF-α and IL-1β the level of induction was nearly comparable to that induced by application of 10 ng of Ec-LPS/ml (
Thus, the data presented in
This Example demonstrates the effects of the holotoxins and their respective B subunits on IL-10 induction. We investigated whether LT-II holotoxins and CT inhibition of proinflammatory cytokine induction by Ec-LPS or other bacterial stimuli, such as Pg-LPS and FimA (
Further analysis of the data indicated that there was a correlation between the ability of the holotoxins to upregulate IL-10 (
This Example provides an analysis of the role of IL-10 in holotoxin-mediated TNF-α and IL-8 downregulation in activated cells. To determine whether the downregulatory effects of the holotoxins on TNF-α and IL-8 induction in activated cells were mediated via induction of IL-10, experiments were conducted using a neutralizing MAb to IL-10 (10 μg/ml) obtained from R&D Systems (Minneapolis, Minn.). If the downregulatory effects were caused by IL-10, then addition of the anti-IL-10 MAb to the cell cultures would be expected to reverse the inhibitory effects of LT-IIa, LT-IIb, or CT on production of these proinflammatory cytokines by cells activated with LT-IIb-B. Although anti-IL-10 significantly (P<0.05) counteracted holotoxin-mediated inhibition of TNF-α or IL-8 induction by LT-IIb-B, the reversal was only partial (Table 1). The use of a higher concentration of anti-IL-10 (20 μg/ml) did not further enhance the reversal effect (data not shown). Similarly, anti-IL-10 only partially reversed holotoxin-mediated inhibition of FimA-induced TNF-α (data not shown). Thus, these data suggest that endogenous production of IL-10 cannot adequately account for the ability of the holotoxins to downregulate proinflammatory cytokine induction. Nonetheless, the data demonstrate that the holotoxins interfere with pro-inflammatory immunological responses.
αTHP-1 cells were pretreated for 1 h with holotoxins (either LT-IIa, LT-IIb, or CT; all at 2 μg/ml) in the absence or presence of anti-IL-10 MAb (10 μg/ml). The cells were then stimulated with LT-IIbB (2 μg/ml). After 16 h, culture supernatants were analyzed by ELISA for TNF-α and IL-8 release.
*Statistically significant (P < 0.05) inhibition of LT-IIbB-induced cytokine release by holotoxin.
**Statistically significant (P < 0.05) counteraction of the holotoxin inhibitory effect on LT-IIbB-induced cytokine release. Substitution of isotype-matched control for anti-IL-10 was not statistically different from pretreatment with holotoxin alone (data not shown).
This Example demonstrates the effects of LT-II and CT holotoxins on NF-κB activation. Because NF-κB plays a central role in the activation of genes encoding proinflammatory cytokines (Akira, S., 2001, Adv. Immunol., 78:1-56), it was determined whether LT-II enterotoxins and CT downregulate cytokine induction in LT-IIb-B-stimulated cells by interfering with NF-κB activation. Although both p50 and p65 subunits of NF-κB bind target DNA upon NF-κB activation, the p65 subunit was selected for examination because p65 is the transactivating subunit of heterodimeric (p50/p65) NF-κB. THP-1 cells were treated with LT-IIb-B, and the level of activation of NF-κB was measured as described below. FimA was used in a parallel experiment as a positive control for NF-κB p65 activation (Hajishengallis, G., et al., 2004, Infect. Immun. 72:1188-1191), and IL-10 (10 ng/ml) was used as a positive control for inhibition of NF-κB activation (Raychaudhuri, B., et al., 2000, Cytokine 12:1348-1355, Schottelius, A. J. G., et al., 1999, J. Biol. Chem. 274:31868-31874). Briefly, NF-κB activation in THP-1 cells was determined by means of an NF-κB p65 ELISA-based transcription factor assay kit (Active Motif, Carlsbad, Calif.) (Hajishengallis, G., et al., 2004, Infect. Immun. 72:1188-1191, Hajishengallis, G., et al., 2002, Infect. Immun. 70:6658-6664). The detecting antibody used in this ELISA recognizes an epitope on the p65 subunit of NF-κB that is accessible only when NF-κB is activated and bound to its target DNA (containing the NF-κB consensus binding site) attached to 96-well plates. The assay was used to determine LT-IIb-B-induced NF-κB activation and its regulation by holotoxins. Specifically, differentiated THP-1 cells were preincubated at 37° C. for 1 h with culture medium or in the presence of holotoxins as potential downregulators of NF-κB activation. Cells were subsequently stimulated for 90 min with LT-IIb-B. IL-10 was used as a positive control for downregulation of NF-κB activation while FimA was utilized as a positive control for NF-κB activation. Extract preparation and ELISA to detect NF-κB p65 were performed according to the manufacturer's protocols. The optimal time of stimulation and amount of total protein (7.5 μg) used in the ELISA were determined empirically in preliminary experiments.
Results from these experiments indicate that LT-IIb-B did activate NF-κB p65 (Table 2), thus presenting a plausible mechanism for proinflammatory cytokine induction by LT-IIb-B. Boiling of LT-IIb-B at a relatively dilute concentration (<10 μg/ml) to facilitate disassembly of the unusually stable pentameric structure was correlated with a loss in the molecule's ability to activate NF-κB (Table 2).
αTHP-1 cells were preincubated for 1 h with IL-10 (10 ng/ml) or holotoxins (either LT-IIa, LT-IIb, or CT; all at 2 μg/ml) prior to stimulation with LT-IIbB (2 μg/ml) or FimA (1 μg/ml), which was used as a positive control for NF-κB activation. Boiled LT-IIbB
served as a negative control for stimulus, whereas boiled LT-IIb served as a negative control for pretreatment. After 90 min of stimulation, cellular extracts were analyzed for NF-κB p65 activation by using an ELISA-based kit (Active Motif). After 16 h, culture supernatants
were analyzed by ELISA for TNF-α and IL-1β release. Data shown are means ± standard deviations, n = 3.
*Statistically significant (P < 0.05) differences between non-pretreated controls and groups pretreated with IL-10 or holotoxin. OD450, optical density at 450 mm.
This result excludes the possibility that the activation effect was mediated by incidental heat-stable contaminants in the preparation of purified LT-IIb-B. IL-10 significantly (P<0.05) inhibited both LT-IIb-B-mediated activation of NF-κB and the release of TNF-α and IL-1β (Table 2). LT-IIa, LT-IIb, and CT also partially inhibited LT-IIb-B-mediated activation of NF-κB (P<0.05), although the effect was lost when the holotoxins were denatured by boiling (Table 2). It is most likely that the inhibitory effect of the holotoxins on NF-κB activation is IL-10-independent; inhibition of NF-κB p65 activation occurred within 90 min of cellular activation (Table 2), i.e., earlier than release of IL-10 in our experimental system (IL-10 was undetectable after only 2 h of cellular stimulation with LT-IIb-B in the presence or absence of the holotoxins; data not shown). As observed with LT-IIb-B, we found that the holotoxins and IL-10 also regulated FimA-mediated NF-κB activation and cytokine release (Table 2). Thus, this Example demonstrates an intact holotoxin can antagonize the effects of its isolated B pentamer.
EXAMPLE 8 For this and the following Examples, the construction of His-tagged versions of reduced ganglioside binding mutants of LT-IIa-B with a Thr to Ile substitution at position 34 (termed “LT-IIa-B(T34I)”) and of LT-IIb-B with a Thr to Ile substitution at position 13 (termed “LT-IIb-B(T34I)” was performed essentially as described in Example 1, but using pTDC400/T34I (Connell, T., et al., 1992, Infect. Immun. 60:63-70) and pTDC700/T13I, Connell, T. D., et al., 1995, Mol. Microbiol. 16:21-31), respectively, as the starting materials. The resulting plasmids encoding for LT-IIa-B(T34I) and LT-IIb-B(T13I) were denoted pHN22 and pHN19, respectively. The purity of the holotoxins and their respective B subunits was confirmed as specified in Example 1. A representative sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis separation of the purified holotoxins and their B subunits is shown in
The amino acid sequence of LT-IIb-B(T13I) polypeptide has the sequence shown as SEQ ID NO:11. The complete sequence of LT-IIb and the demonstration that this mutant is non-toxic is available in Connell et al., 1995, Molecular Microbiology, 16:21-31, incorporated herein by reference.
The amino acid sequence of the LT-IIa-B(T34I) mutant is shown as SEQ ID NO:12. The complete sequence of the LT-IIa polypeptide is available as Accession no. M17894 and the complete sequence of the LT-IIb polypeptide is available as Accession no. M28523.
EXAMPLE 9This Example demonstrates that TLR2 is involved in B pentamer-induced cytokine release in THP-1 cells. Several microbial proteins appear to display molecular patterns that can activate cells through “Toll-Like Receptors” (TLRs). Whether LT-II B pentamer-induced cellular activation is dependent on TLRs was addressed in cytokine induction assays using THP-1 cells and anti-TLR mAbs. For these experiments, pentameric B subunits of LT-II or CT were used at 2 μg/ml unless otherwise stated. Stimulation was performed in the absence or presence of blocking monoclonal antibodies (mAbs) to TLR2 (TL2.1), TLR4 (HTA125), or immunoglobulin (Ig) isotype-matched (IgG2a) control (e-Bioscience, San Diego, Calif.). None of the molecules was found to affect cell viability as determined by trypan blue exclusion. Culture supernatants were collected after 16-h incubation and stored at −80° C. until assayed for cytokine content using ELISA kits (from eBioscience or Cell Sciences, Canton, Mass.). Similar cell culture procedures were followed to assess cytokine induction (using eBioscience ELISA kits) in mouse peritoneal macrophages from C57BL/6 wild-type mice or mice deficient in TLR2 (Takeuchi, O., et al., 1999, Immunity 11:443-451) or TLR4 (Hoshino, K., et al., 1999, J. Immunol. 162:3749-3752) that have been 9-fold backcrossed on the C57BL/6 genetic background.
We found that IL-8 induction by LT-IIa-B, LT-IIb-B, or CTB was partially but significantly (P<0.05) inhibited by a mAb to TLR2 (
Induction of IL-8 release in THP-1 cells by 2 μg/ml of LT-IIa-B (4752±611 pg/ml), LT-IIb-B (28530±4367 pg/ml), or CTB (704±84 pg/ml), was unaffected in the presence of 10 μg/ml polymyxin B (corresponding IL-8 responses: 4459±489 pg/ml; 30530±3005 pg/ml; 789±92 pg/ml, respectively) but was abrogated upon boiling of the B pentamers (corresponding IL-8 responses: 147±45 pg/ml; 132±64 pg/ml; 108±28 pg/ml, respectively). Conversely, when THP-1 cells were activated by 0.2 μg/ml of E. coli LPS, the induced IL-8 release (34839±3187 pg/ml) was inhibited by polymyxin B (3098±618 pg/ml) but not by boiling the LPS (37122±5890 pg/ml). These findings verify that activation of the cells by B pentamers was not attributable to contamination with LPS or other heat-stable contaminants.
EXAMPLE 10 This Example demonstrates that LT-II-B pentamers activate TLR1/TLR2-transfected HEK 293 cells. To further demonstrate TLR2 involvement in B pentamer-induced cellular activation, we used HEK 293 cells transiently cotransfected with cDNAs encoding TLR2 with either TLR1 or TLR6, both of which have been shown to cooperate with TLR2 to mediate signaling (Mielke, P. W., Jr., et al., 1982, Commun. Statist.—Theory Meth. 11: 1427-1437). For these experiments, HEK 293 cells were plated in 24-well tissue culture plates (5×104 cells per well) in 0.5 ml complete RPMI (as above except that 2-mercaptoethanol was not included). The cells were incubated for 16-20 hrs after plating at 37° C. in 5% CO2 to about 50% confluency. Each well was transfected with 25 ng pRLnull renilla luciferase reporter (Promega, Madison Wis.), 75 ng NF-κB firefly luciferase reporter and one of the following: empty FLAG-CMV vector alone (100 ng), TLR2 (10 ng) and TLR1 (90 ng), or TLR2 (10 ng) and TLR 6 (90 ng). All the TLRs are N-terminal FLAG tagged derivatives of the human receptors. The DNA mixture was mixed with 5 μl CaCl2 (2.5 M) and sterile water to a volume of 50 μl, after which 50 μl of 2× HEPES-buffered saline was added. The DNA precipitate was then added dropwise to the cells, incubated for 6 hrs at 37° C. in 5% CO2 after which the media were replaced. Two days after transfection, the cells were stimulated with either no agonist, 20 ng/ml Pam3Cys-Ser-Lys4 lipopeptide (Pam3Cys; EMC Microcollections, Tuebingen, Germany) or 2 μg/ml of holotoxin or B pentamer preparations. After 16 hrs of stimulation, the media were aspirated and 50 μl of Passive Lysis Buffer (Promega) was added to the plates which were incubated with rocking for 15 minutes at room temperature. Lysates were transferred to a 96-well plate and 10 μl of each lysate was evaluated for luciferase activity using the Dual-Luciferase Reporter Assay System. (Promega). Each firefly luciferase value was divided by the Renilla value to correct for transfection efficiency. All corrected values were normalized to that of no agonist whose value was taken as 1. A non-parametric procedure was used to analyze the data from the luciferase gene reporter assays (
Accordingly, HEK 293 cells transfected with TLRs or “empty” control vector were stimulated with LT-IIa-B, LT-IIb-B, CTB, or their respective holotoxins. Pam3Cys, a synthetic TLR2 agonist (Hertz, C. J., et al., 2001, J. Immunol. 166:2444-2450), was used as a positive control. All cotransfections included a cDNA encoding firefly luciferase driven by a NF-κB-dependent promoter in order to monitor cellular activation. We found that, besides Pam3Cys, only LT-IIa-B and LT-IIb-B induced significant (P<0.05) cellular activation upon transfection with TLRs (
This Example demonstrates that TLR2 is likely required for LT-II B pentamer-induced cytokine release in mouse macrophages. We evaluated the ability of LT-IIa-B or LT-IIb-B to induce cytokine release in TLR2-deficient macrophages compared with wild-type or TLR4-deficient cells. To elicit peritoneal macrophages, mice were injected with 3 to 4 ml of sterile 3% thioglycollate and cells were harvested after 5 days by flushing the peritoneal cavity with 10 ml of ice-cold PBS four times. Isolated cells were then subjected to density gradient centrifugation (Histopaque 1.083) to remove dead cells and red blood cell contamination. Cells were then washed three times with PBS and re-suspended in complete RPMI medium at 1×106/ml. Known TLR agonists (Pam3Cys, TLR2; E. coli LPS, TLR4) were used as positive or negative controls. All control TLR agonists and LT-II B pentamers induced release of TNF-α (
This Example demonstrates that LT-II B pentamers likely require different ganglioside binding for cellular activation.
Since TLRs often require co-operation with other pattern-recognition [receptors (PRRs) to mediate cellular activation, we determined whether ganglioside binding may be important for the ability of LT-IIa-B or LT-IIb-B to induce TLR2-dependent activation of THP-1 cells. For this purpose we used two mutants, LT-IIa-B(T34I) and LT-IIb-B(T13I), which show no detectable binding to any gangliosides as tested herein, such as GD1a, GD1b, GT1b, GQ1b, GM1, GM2, or GM3 (Connell, T., et al., 1992, Infect. Immun. 60:63-70, (Connell, T. D., et al., 1995, Mol. Microbiol. 16:21-31).
Surprisingly, we found that LT-IIa-B(T34I)was even more effective than the wild-type molecule in inducing cytokine release or NF-κB p65 activation (Table 3; NF-κB activation experiments performed as described in Example 7). Therefore, whereas TLR2 appears to be important for cellular activation by LT-IIa-B (Table 3), gangliosides (at least the ones mentioned above that include those which may be important for LT-IIa toxicity) do not play a role in this regard. On the other hand, the LT-IIb-B(T13I) mutant did not retain any of the proinflammatory activity (cytokine induction or NF-κB p65 activation; Table 3) of the wild-type molecule. Therefore the high-affinity receptor of LT-IIb-B, GD1a, may also be required also for the ability of this molecule to activate THP-1 cells in a TLR2-dependent mode (Table 3).
*THP-1 cells were pretreated for 30 min with anti-TLR2 MAb (10 μg/ml) or medium only prior to stimulation with LT-II B pentamers or nonbinding mutants thereof (all at 2 μg/ml). Induction of cytokine release in culture supernatants, collected 16 h after stimulation, was evaluated by ELISA. In a similar experiment, cellular extracts were prepared
This Example demonstrates the adjuvant activities of wild type and mutant LT-IIa and LT-IIb holotoxins and their respective wild type B pentamers in a mouse mucosal inmmunization model. Mice were intranasally administered LT-II holotoxins or isolated B pentamers as indicated in
This Example demonstrates the level of cAMP activity induced by holotoxins and B pentamers in RAW264.7 macrophage cells. To conduct these experiments, RAW264.7 macrophage cells (5×107) were treated for 6 hrs with 1 microgram of either holotoxin or B pentamer. The amount of cAMP in the treated cells was measured by a competition ELISA (Cayman Chemicals, Ann Arbor, Mich.). As can be seen from the results depicted in
This Example provides an evaluation of ganglioside-binding activity and adjuvant activity for wild type LT-IIa or LT-IIb holotoxins and for their respective single-point substitution mutants (LT-IIa(T34I) and LT-IIb(T13I). Engineering and purification of His-tagged wild type and mutant LT-II holotoxins for this Example were performed essentially as described in Examples 1 and 8 herein, respectively.
Ganglioside-dependent ELISA. Binding of LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I) to their ganglioside receptors were measured as previously described (Connell, T., et al., 1992, Infect. Immun. 60:63-70, Connell, T. D., et al., 1995, Mol. Microbiol. 16:21-31) with some modifications. Briefly, polyvinyl 96-well ELISA plates were coated overnight at 4° C. with 10 ng GT1b, GQ1b, GM2, GM3, GM1, GD1a, GD1b, GD2, or with a ganglioside mixture (Matreya, State College, PA and Sigma Chemical Company, St. Louis, Mo.), or with 3.0 μg/ml goat anti-LT-IIa or goat anti-LI-IIb antibodies. After washing and blocking of non-specific binding with 10% horse serum, 50 μl of 1.0 μg/ml of LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I) was added to wells and plates were incubated for 3 hours at 37° C. Unbound enterotoxins were washed away and 50 μl of rabbit anti-LT-IIa or LT-IIb (diluted 1:5000 in PBS+10% horse serum) were added to the wells. Plates were incubated for another two hours at 37° C. and washed to remove unbound antibodies. Fifty μl of 1.0 μg/ml of alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody were added to each well. Plates were incubated for one hour at 37° C. after which wells were washed and immediately developed with nitrophenyl phosphate (Amresco, Solon, Ohio) diluted in diethanolamine buffer (100 ml diethanolamine, 1 mM MgCl2, deionized H2O to 1 liter; pH 9.8). Color reactions were terminated by adding 50 μl 2.0M NaOH to each well. Optical density of the color reaction was measured at 405 nm.
Toxicity bioassay. The toxicity of purified enterotoxins was measured using Y1 adrenal cells (ATCC CCL-79), a cell line which is acutely sensitive to heat-labile enterotoxins. Briefly, mouse Y1 adrenal cells were cultured to 50% confluence in 96 well tissue culture plates in F-12 medium supplemented with 30% horse serum and 10% fetal bovine serum at 37° C. and in an atmosphere of 5% CO2. One microgram of CT, LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I) per well was added to the Y1 cell cultures and diluted in a 2-fold dilution series across the plate. Plates were incubated at 37° C. in an atmosphere of 5% CO2 and examined for 48 hrs to monitor rounding of cells which is an indicator of toxicity. One unit of toxicity is defined as the smallest concentration of enterotoxin that induces rounding of 75 to 100% of the cultured mouse Y1 adrenal cells.
Animals and immunizations. Female BALB/c mice, 11 to 12 weeks of age, were immunized by the intranasal (i.n.) route. Groups of 8 mice were immunized three times at 10-day intervals with AgI/II (10 μg) alone or with AgI/II in combination with 1 μg of CT, LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I). Immunizations were administered in a standardized volume of 10 μl, applied slowly to both external nares. At day 203 after initial immunization all groups were re-immunized i.n. with 5 μg of AgI/II alone. All animal experiments were approved by the Institutional Animal Care and Use Committee at the State University of New York at Buffalo.
Collection of secretions and sera. Samples of serum, saliva, and vaginal washes were collected from individual mice 2 days before the initial immunization (day 0) and at 18, 28, 42, 60, and 175 days after the primary immunization. Saliva samples were collected with a micropipetter after stimulation of salivary flow by injecting each mouse intraperitoneally with 5 μg of carbachol (Sigma). Vaginal washes were collected by flushing the vaginal vault three times with 50 μl of sterile PBS. Serum samples were obtained following centrifugation of blood collected from the tail vein by use of a calibrated capillary tube. Mice were sacrificed at day 217 and blood was collected after cardiac puncture using 20-gauge syringe needles. Mucosal secretions and serum samples were stored at −70° C. until assayed for antibody activity.
Antibody analysis. Levels of isotype-specific antibodies in saliva, sera, and vaginal washes were measured by enzyme-linked immunosorbent assay (ELISA). Polystyrene microtiter plates (96-well; Nunc, Roskilde, Denmark) were coated overnight at 4° C. with AgI/II (5 μg/ml), LT-IIa (3 μg/ml), LT-IIb (3 μg/ml), or CT (3 μg/ml). To determine total immunoglobulin (Ig) isotype concentrations, plates were coated with goat anti-mouse Ig isotype-specific antibodies (Southern Biotechnology Associates, Birmingham, Ala.). Serial twofold dilutions of serum or secretion samples were added in duplicate, and plates were incubated overnight at 4° C. Plates were washed with PBS containing 0.1% Tween-20 (PBS-Tw) and incubated at RT with the appropriate alkaline phosphatase-conjugated goat anti-mouse Ig isotype-specific antibodies (Southern Biotechnology). Plates were washed and developed with nitrophenyl phosphate, as described previously. Concentrations of antibodies and total IgA levels were calculated by interpolation of calibration curves generated by using a mouse Ig reference serum (ICN Biomedicals, Aurora, Ohio). Mucosal IgA responses are reported as the percentage of specific antibody IgA in total IgA to compensate for variations arising from salivary flow rate and dilution of secretions. All enterotoxins were able to induce anti-enterotoxin serum IgG. LT-IIa(T34I) induced lower level of serum IgG than its wild type while LT-IIb(T13I) induced equivalent level of serum IgG as its wild type (data not shown).
Isolation of lymphoid cells. Superficial cervical lymph nodes (CLN) were excised as previously described (Martin, M., et al., 2000, Infect. Immun. 68:281-287). CLN and spleens were teased apart with syringe pistons, dispersed through a 70-μm nylon-mesh screen, and passed twice through 26 gauge syringe needles to obtain single-cell suspensions. Cell suspensions were filtered through nylon mesh to remove tissue debris and centrifuged through Ficoll-Hypaque 1083 (Sigma) to remove erythrocytes and dead cells. All preparations were washed twice and suspended in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). Total cell yield and viability were enumerated in a hemacytometer using trypan blue (Sigma) staining.
Cytokine assays. Spleen and CLN lymphoid cells were plated in triplicates at 5×105 cells per well in flat-bottomed, 96-well tissue culture plates (Nunc), and cultured for 4 days in the presence of concanavalin A (2.5 μg/ml), AgI/II (5 μg/ml) or in the absence of stimulus. Supernatants were collected after centrifugation and stored at −70° C. until assayed for the presence of cytokines. The levels of interleukin-4 (IL-4) and gamma interferon (IFN-γ) in culture supernatants were determined by a cytokine-specific ELISA according to the manufacturer's protocol (Pharmingen, San Diego, Calif.). Briefly, 96-well culture plates were coated with monoclonal anti-IL-4 or anti-IFN-γ (2 μg/ml) and incubated overnight at 4° C. Plates were washed with PBS-Tween and blocked to limit nonspecific binding with 10% FBS in PBS for 1 h at RT. After washing the plates, supernatants were serially diluted in 10% FBS in PBS and added to the wells. A standard curve was generated by using serial dilutions of recombinant IL-4 (500 pg/ml) or IFN-γ (2,000 pg/ml). All serial dilutions were incubated at 37° C. for three hrs followed by washing with PBS-Tween. Secondary antibodies consisted of peroxidase-labeled anti-IL-4 or biotinylated anti-IFN-γ. In assays using biotinylated antibodies, a 1:1,000 dilution of horseradish peroxidase-conjugated streptavidin in 10% FBS in PBS was added to the appropriate wells. After incubation at RT for 2 hrs, reactions were developed for 20 min with o-phenylenediamine-H2O2 substrate and terminated by addition of 1.0 M H2SO4. The color reaction was measured at 490 nm.
Binding of enterotoxins to CLN lymphoid cells. 106 cells obtained from CLN of naïve mice were treated in vitro with 1.0 μg of LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I). After incubation on ice for 10 minutes, cells were washed and subsequently incubated on ice for 10 minutes with a pre-titrated concentration of polyclonal rabbit antibody to LT-IIa or LT-IIb. After washing, cells were treated with phycoerythrin (PE)-conjugated goat anti-rabbit IgG (0.5 μg/ml) and with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody to CD3, CD4, CD8, B220, or CD11b. After incubation for 10 minutes on ice, cells were washed and then incubated with 1.0 μg/ml of propidium iodide. CD16/CD32 antibodies were used to block Fc receptor following the manufacturer's instructions. Enterotoxin-binding mutants (1.0 μg), isotype-matched fluorochrome-labeled antibodies, and specific anti-enterotoxin rabbit sera were used as controls to set detection limits. Data acquisition and analysis were performed using a FACScalibur flow cytometer (Beckton-Dickinson, Franklin Lakes, N.J.) and the CellQuest software (Beckton-Dickinson).
Detection of Adenosine 3═,5′ cyclic monophosphate (cAMP). cAMP production was measured in mouse macrophage RAW264.7 cells (ATCC TIB-71) as a relevant lymphoid cell type. Briefly, mouse macrophage RAW264.7 cells (5×107 per well ) were cultured in triplicates for 24 hrs in 24-well tissue culture plates at 37° C. and in atmosphere of 5% CO2 in Dulbecco's Modified Eagle medium supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 10% fetal bovine serum. Culture medium was removed and replaced with fresh culture medium with or without 1.0 μg/ml CT, LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I). After incubation at 37° C. for 4 hrs, enterotoxin-treated cells were extracted with 200 μl of 0.1 M HCl for 20 minutes at RT, scraped from the wells, and centrifuged to clear the extracts of cells and cell debris. Levels of cAMP in the extracts were measured twice using a cAMP EIA·kit (Cayman Chemical Co., Ann Arbor, Mich.) according to the manufacture's protocols.
Statistical analysis. Analysis of variance (ANOVA) and the Tukey multiple-comparison test were used for multiple comparisons. Unpaired t tests with Welch correction were performed to analyze differences between two groups. Statistical analyses were performed using InStat (GraphPad, San Diego, Calif.). Statistical differences were considered significant at the P<0.05 level.
Purification of wt and mutant LT-IIa and LT-IIb. To facilitate their purification, recombinant LT-IIa, LT-IIa(T34I), LT-IIb, and LT-IIb(T13I) holotoxins were engineered with His-tags fused to the carboxyl end of the B pentamers. His-tagged holotoxins were purified from periplasmic extracts of recombinant E. coli using a two-step chromatographic protocol. In the first step, holotoxins and B pentamers were isolated from periplasmic extracts using nickel affinity chromatography. Holotoxins were separated from the contaminating B pentamers by subsequent gel-filtration chromatography. Recombinant wt and mutant holotoxins were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using polyclonal antibodies directed toward LT-IIa or LT-IIb to demonstrate that each enterotoxin was purified to apparent homogeneity (
Binding of wt and mutant LT-IIa and LT-IIb to gangliosides. Reduction of binding of LT-IIa(T34I) and LT-IIb(T 13I) to gangliosides was originally defined using periplasmic extracts from recombinant strains of E. coli as crude sources of the mutant enterotoxins (Fukuta, S., et al., 1988, Infect. Immun. 56:1748-1753). To confirm that the ganglioside-binding activities of the purified mutant enterotoxins were equivalent to those of the mutant enterotoxins in the crude extracts, binding of the purified wt and mutant enterotoxins for various gangliosides was measured by ganglioside-specific ELISA (Connell, T., et al., 1992, Infect. Immun. 60:63-70, Connell, T. D., et al., 1995, Mol. Microbiol. 16:21-31) (
Toxicity of LT-IIa(T34I) and LT-IIb(T13I). Prior results using crude periplasmic extracts from recombinants expressing LT-IIa(T34I) and LT-IIb(T13I) indicated that LT-IIa(T34I) and LT-IIb(T13I) were severely attenuated in toxicity (Connell, T., et al., 1992, Infect. Immun. 60:63-70, Connell, T. D., et al., 1995, Mol. Microbiol. 16:21-31). To confirm those results using purified wild type and mutant holotoxins, Y1 adrenal cell toxicity assays were repeated. Comparisons of the toxicities revealed that CT was the most toxic of the five enterotoxins. Only 0.49 ng of CT was sufficient to induce rounding of 100% of Y1 adrenal cells within a test well. LT-IIa was 16-fold less toxic, requiring 15.65 ng of enterotoxin to cause the same effect. LT-IIa(T34I) exhibited no detectible toxic activity at levels up to 1.0 μg of enterotoxin. Only after 24 hours of incubation with LT-IIa(T34I) was any toxicity detected, i.e. 10% of the cells in the well containing 1.0 μg and 0.5 μg of enterotoxin demonstrating a “rounding” morphology. Y1 adrenal cells had to be incubated with 8-fold the amount of LT-IIb (0.49 ng vs 3.91 ng) to elicit the same degree of toxicity for Y1 adrenal cells as by CT. In comparison, LT-IIb(T13I) was 256-fold less toxic than LT-IIb. In conclusion, the LT-IIa and LT-IIb were significantly less toxic than CT by the Y1 adrenal cell bioassay, and each of the respective mutant enterotoxin was significantly less toxic than its wt parent enterotoxin.
Mucosal adjuvant activities of LT-IIa(T34I) and LT-IIb(T13I). To compare the adjuvant activities of the mutant enterotoxins with the wt enterotoxins, mice were intranasally immunized with AgI/II (Russell, M. W., et al., 1980, Infect. Immun. 28:486-493), in the presence or absence of LT-IIa or LT-IIb. CT was utilized as an external control, as the mucosal adjuvant activities of this enterotoxin for AgI/II have been well-established (Martin, M., et al., 2000, Infect. Immun. 68:281-287, Wu, H. Y., et al., 1998, Vaccine 16:286-292).
Initial immunizations were followed by booster immunizations at day 10 and at day 20. Saliva and vaginal secretions, obtained at intervals up to 175 days after the initial immunization, were analyzed for AgI/II-specific IgA antibodies as a measure of mucosal adjuvant activity of the enterotoxins.
Immunization with AgI/II alone did not elicit a strong salivary IgA response to the antigen (
When the salivary anti-AgI/II IgA responses of mice immunized with AgI/II+LT-IIa(T34I) were measured, it was found that the mutant enterotoxin was capable of inducing higher mean value of anti-AgI/II IgA antibodies at day 28, but those values were not statistically significant (P>0.05), from mice immunized with AgI/II alone due to high variation among mice (
LT-IIa and LT-IIb when used as intranasal adjuvants were also capable of inducing strong immune responses to a co-administered antigen at distal mucosa (Martin, M., et al., 2000, Infect. Immun. 68:281-287). To determine whether mucosal adjuvant responses were potentiated at distal sites in these experiments, levels of AgI/II-specific IgA was measured in samples taken from the vaginal mucosa (
From these results, it was clear that the mucosal adjuvant activity of LT-IIa(T34I) was diminished by reduction of binding affinity for its known ganglioside receptors (e.g. GD1b, GM1, GT1b, GQ1b, GD2, GD1a and GM3). In the case of LT-IIb(T13I), however, the mutation had little or no effect on mucosal adjuvant activity. The mucosal adjuvant activities of LT-IIb(T13I) for inducing antigen-specific IgA, surprisingly, were indistinguishable from the mucosal adjuvant activities of wt LT-IIb.
Systemic adjuvant activity of LT-IIa(T34I) and LT-IIb(T13I). Intranasal administration of LT-IIa, LT-IIb and CT also induces strong circulating antibody responses to co-administered antigens (Connell, T. D., et al., 1998, Immunology Letters 62:117-120, Martin, M., et al., 2000, Infect. Immun. 68:281-287). To examine whether mucosal immunomodulatory activities of LT-IIa(T34I) and LT-IIb(T13I) had the capacity to potentiate serum antibody responses, antigen-specific IgA and antigen-specific IgG were measured in serum samples taken at various time points from mice intranasally immunized with AgI/II in the presence and absence of mutant or wt enterotoxins.
As expected, both LT-IIa and LT-IIb potentiated anti-AgI/II serum IgA after intranasal administration with AgI/II (
At all time points tested, serum IgG responses to AgI/II were also elevated in mice immunized with AgI/II+LT-IIa (P<0.05), AgI/II+LT-IIb (P<0.001), and AgI/II+LT-IIb(T13I) (P<0.001) compared to mice immunized with AgI/II alone (
Serum IgG subclasses responses. Based on IgG subclass distribution, LT-IIb stimulates a more balanced T helper 1 (Th1)/T helper 2 (Th2) immune response than either CT or LT-IIa (Martin, M., et al., 2000, Infect. Immun. 68:281-287). To determine if the mutant enterotoxins stimulated IgG subclass distribution similar or different from those stimulated by their wt parent enterotoxins, the concentrations of AgI/II-specific IgG1, IgG2a, and IgG2b were determined in the serum obtained at day 28. Immunization with AgI/II alone induced low levels of IgG1, IgG2a, IgG2b (
Cytokine production. To complement the IgG subclass distribution experiments, expression patterns for IFN-γ and IL-4 were measured in lymphoid cells obtained from the draining superficial cervical lymph nodes (CLN) and from the spleens of immunized mice after in vitro AgI/II stimulation (
Binding of wt and mutant LT-IIa and LT-IIb to lymphocytes. In vitro binding experiments revealed that LT-IIb(T13I) had little or no detectable binding affinity for ganglioside receptors. Furthermore, exhibits extremely low toxicity for Y1 adrenal cells (Connell, T. D., et al., 1995, Mol. Microbiol. 16:21-31), indicating that the mutant enterotoxin is incapable of inducing production of cAMP, a potent intracellular messenger for a variety of metabolic processes. Thus, we tested whether LT-IIb(T13I) interacts with one or more types of lymphoid cells. To determine whether LT-IIb(T13I) had residual binding affinity for lymphoid cells, cells from the CLN of naïve mice were incubated with wt LT-IIb or with LT-IIb(T13I) and subsequently examined by flow cytometry for bound enterotoxin (
cAMP production in macrophages treated with LT-IIa(T34I) and LT-IIb(T13I). Although LT-IIa(T34I) and LT-IIb(T13I) had no detectable binding in vitro to their major ganglioside receptors (
Claims
1. A method of enhancing an immune response to an antigen in an individual comprising administering to the individual a composition comprising an effective amount of:
- a) an isolated LT-IIb-B pentamer or an isolated LT-IIa-B pentamer; and
- b) the antigen;
- whereby the LT-IIb-B pentamer or the LT-IIa-B pentamer acts as an adjuvant to enhance the immune response to the antigen.
2. The method of claim 1, wherein the LT-IIb-B pentamer is a mutant LT-IIb-B pentamer having a mutation selected from the group consisting of: replacement of threonine by isoleucine, lysine or asparagine at the 13th position; and replacement of threonine by isoleucine, asparagine, arginine, methionine or lysine at the 14th position.
3. The method of claim 2, wherein the mutation of the LT-IIb-B pentamer is a replacement of threonine by isoleucine at the 13th position of the LT-IIb-B pentamer amino acid sequence.
4. The method of claim 1, wherein the LT-IIa-B pentamer is a mutant LT-IIa-B pentamer having mutation selected from the group consisting of: replacement of threonine by isoleucine, proline, glycine, asparagine, leucine or arginine at the 13th position; replacement of threonine by isoleucine, proline, aspartic acid, histidine and asparagine at the 14th position; and replacement of threonine by isoleucine, alkaline, glycine, methionine, histidine, leucine, arginine or glutamine at the 34th position.
5. The method of claim 4, wherein wherein the mutation of the LT-IIa-B pentamer is a replacement of theronine by isoleucine at the 34th position of the LT-IIa-B pentamer amino acid sequence.
6. The method of claim 1, wherein the antigen and the LT-IIb-B pentamer or the LT-IIa-B pentamer are administered mucosally.
7. The method of claim 6, wherein the mucosal administration is selected from the group of routes consisting of intranasal, ocular, gastrointestinal, oral, rectal and genitourinary tract.
8. The method of claim 7, wherein the mucosal administration is intranasal administration.
9. The method of claim 1, wherein the antigen and the LT-IIb-B pentamer or the antigen and the LT-IIa-B pentamer are administered parentally.
10. The method of claim 1, wherein the antigen and the LT-IIb-B pentamer or the the antigen and the LT-IIa-B pentamer are administered via a route selected from the group consisting of intraperitoneal, intravenous, subcutaneous or intramuscular.
11. The method of claim 1, wherein the antigen and the LT-IIb-B pentamer or the antigen and the LT-IIa-B pentamer are administered as a chimeric molecule.
12. The method of claim 1, wherein the antigen and the LT-IIb-B pentamer or the antigen and the LT-IIa-B pentamer are administered as a chemically conjugated molecule.
13. The method of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.
14. The method of claim 1, wherein the enhanced immune response is an enhancement of in the production of IgA antibodies, IgG antibodies, or both.
15. The method of claim 14, wherein the IgA antibodies are mucosal IgA antibodies.
16. The method of claim 14, wherein the IgG antibodies are systemic antibodies.
Type: Application
Filed: Feb 15, 2006
Publication Date: Aug 17, 2006
Inventors: Terry Connell (Williamsville, NY), Michael Russell (East Amherst, NY), Hesham Nawar (Buffalo, NY), Georgios Hajishengallis (Louisville, KY)
Application Number: 11/354,497
International Classification: A61K 39/02 (20060101);
